The goal of this project is to identify endogenous mechanisms controlling SC behavior through molecular manipulation of the AKT-TOR signaling axis in the complexity of the adult body. Stem cell (SC) therapy offers great promise for inducing regeneration of tissue damaged by senescence, cancer or injury. However, the current inability to rationally control the behavior of SCs in vivo severely limits the implementation of effective SC-based treatment. The largely unknown process of SC regulation involves local and systemic signals that coordinate cellular behavior through signaling pathways. Molecular manipulation of cell signaling pathways that integrate local and systemic information offer great potential to control SC behavior in vivo. This approach, however, requires full characterization of SC regulation in their natural environment. This project addresses the fundamental problem of SC-based regeneration and repair using two important elements: 1) a natural sensor: the AKT-TOR axis, an evolutionarily conserved signaling pathway that integrates local and systemic information that is central to cellular and organismal physiology, and 2) a model system: the planarian flatworm Schmidtea mediterranea, which has extraordinary regenerative capacity and abundant adult stem cells (neoblasts) that are accessible for molecular analysis of evolutionarily conserved signaling pathways. The proposed approach provides an elegant, simplified and highly innovative model for systematic investigation of SC transition to differentiation and SC response to injury and physiological cell turnover. This project is based on a comprehensive interdisciplinary approach that studies SCs in the complexity of the whole organism considering both local and systemic endogenous signals that modulate their behavior. It is unique in assessing adult SC regulation during the process of tissue regeneration and cell turnover. The strategy is designed to follow neoblast response in vivo during cell turnover and regeneration after manipulation of endogenous signals that control their behavior. Aim 1 will assess the process of regeneration in the absence of TOR signaling, which is characterized by the absence of regenerative blastema and tissue remodeling. TOR function will be abrogated in intact adults with RNA-interference (RNAi) or specific chemical inhibitors. Aim 2 will determine neoblast contribution to tissue repair mechanisms in the absence of AKT by abrogating AKT function in intact adults with RNAi and treatment with inhibitors. Successful completion of this project will provide mechanistic details of how progenitor cells are instructed to repair complex tissues. These results will provide a systemic analysis in adults of the regulatory effects of the AKT-TOR axis on SCs. This project capitalizes on a cellular signaling pathway that is the focus of many pharmacological approaches to control cancer and degenerative diseases. Anticipated future studies will be translated to vertebrate models and ultimately to clinical regenerative medicine.